Biochemistry, Biophysics, and Structural Biology Commons^™

Role Of E3 Ubiquitin Ligase Ubr5 In B Cell Development And Mantle Cell Lymphoma, Tyler J. Gilbreath

Theses & Dissertations

UBR5 is part of the ubiquitin proteasome system (UPS), most well-known as a degradation and recycling pathway in which proteins are tagged with a chain of ubiquitin protein and degraded by the proteasome. However, the UPS is multifunctional and other important roles include tagging protein with ubiquitin for cell signaling purposes. UBR5 is a homologous to the E6AP C-terminus (HECT) E3 ubiquitin ligase, meaning ubiquitin is transferred to UBR5 on a cysteine residue within the HECT domain and then onto a target substrate. UBR5 has mutations in many types of cancers: breast, ovarian, liver, and lymphomas. Recently published data shows …

Novel Mechanisms Of Protein Kinase C Α Regulation And Function, Xinyue Li

Theses & Dissertations

Protein kinase Cα (PKCα) is a member of the PKC family of serine/threonine kinases, which have been implicated in regulation of many cellular processes, including cell proliferation, differentiation, survival, and transformation. A large body of evidence from the Black laboratory and others support an anti-proliferative function of PKCα in normal epithelial tissues, including the intestinal mucosa and endometrial epithelium. PKCα is also tumor suppressive in epithelial cancers, such as colorectal cancer (CRC) and endometrial cancer (EC). However, a major obstacle to harnessing the tumor suppressive functions of PKCα to benefit patients is the widespread loss of PKCα expression in tumors. …

Metoprolol Disrupts Sterol Biosynthesis Through Inhibition Of 7-Dehydrocholesterol Reductase (Dhcr7), Luke B. Allen

Theses & Dissertations

Cholesterol is essential for life. It is particularly important in the brain as it relies on de novo synthesis of cholesterol following the formation of the blood brain barrier (BBB). As such, disrupting sterol biosynthesis during neurodevelopment can have devastating outcomes. The most common post-lanosterol sterol biosynthesis disorder, Smith-Lemli-Opitz Syndrome, arises from a faulty DHCR7 enzyme. DHCR7 has also been shown to be inhibited by several psychotropic medications. Here we assess six beta-blockers and their effects on sterol biosynthesis in vitro. Two beta-blockers, metoprolol and nebivolol strongly inhibit DHCR7 in four separate in vitro models of both mouse and …

Ewing Sarcoma Transcriptome: Identification Of Foxn3 As A Novel Target Of Ews-Fli1, Natasha Kreiling

Theses & Dissertations

Ewing Sarcoma is an aggressive cancer characterized by a fusion gene formed from a chromosomal translocation between a RNA binding protein and a transcription factor. The most common fusion gene is EWS-FLI1 a potent oncogenic transcription factor responsible for initiating genome instability. EWS-FLI1 promotes tumor growth by dysregulating several genes and pathways. We have identified FOXN3, an important gene in cell cycle control and development, as being suppressed by EWS-FLI1. In the future, a better understanding of FOXN3 and its ability to act as a tumor suppressor in EWS will unravel novel mechanisms of EWS pathogenesis, potential biomarker and drug …

Dysregulation Of Mir-10a Promotes Cancer Features In Cholangiocarcinoma, Matthieu Spriet

Theses & Dissertations

Cholangiocarcinoma is a primary liver cancer of the bile duct epithelium that exhibits microRNA-mediated control of tumor cell signaling. Strides toward new treatment rest on a better defining of cholangiocarcinoma tumor biology including the RNA-based layer of regulation. Additionally, there is a gap in knowledge on microRNA expression in human tissue. While there is RNA-seq data of microRNA expression in tissue, it does not differentiate between cell types, thus leaving unanswered questions about cell specific microRNA biology and expression.

Here, we identify miR-10a as an oncogenic microRNA acting through MAPK signaling. Using cholangiocarcinoma cell lines, we determined miR-10a is an …

Neuropilin-2 In Advanced Prostate Cancer And Its Targeting With Novel Small Molecule Inhibitors, Ridwan Islam

Theses & Dissertations

Despite the development of new therapies, clinical management of advanced metastatic castrationresistant prostate cancer (mCRPC) still remains challenging owing to the emergence of therapy resistance. Resistance to the therapies is driven by both AR and non-AR pathways. Non-AR pathway is characterized by a lineage switch that leads to the development of neuroendocrine-like (NE-like) prostate cancer (PCa), which has a fulminant clinical course due to the lack of effective treatment strategy. Therefore, novel molecular targets need to be identified to develop effective therapeutic regimen to improve the outcomes in advanced PCa patients including NE-like PCa. Recent investigation on advanced PCa patients …

Innate Immunity In The Pathobiology And Treatment Of Infectious And Neurodegenerative Diseases, Mai Mostafa

Theses & Dissertations

Mononuclear phagocytes (MPs; monocytes, macrophages, and dendritic cells) are the governors of innate immunity which is the body’s first line of defense against microbial pathogens. They act beneficial or detrimental. They are crucial for an effective non-specific immune response to invading pathogens by engulfing, destroying, then eliciting an adaptive specific immune response. Given their pivotal functions in the host immune defense, studying MP immune responses in disease is paramount important for understanding disease pathobiology and uncovering therapeutic strategies.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the driver of acute respiratory distress syndrome (ARDS) in coronavirus disease 2019 (COVID-19) amongst …

Mechanisms Of Sorting And Fission At The Endosomes, Kanika Dhawan

Theses & Dissertations

Endocytic trafficking is a fundamental cellular process that regulates the transport of lipids and proteins. Our lab focuses on the intracellular trafficking of receptors involved in cellular processes such as cell division, migration, and proliferation. Accordingly, the regulation of these trafficking pathways is tightly controlled, involving a complex series of events, of which a key step is the endosomal fission. Perturbations in the endosomal network can eventually lead to impaired receptor recycling to the plasma membrane (PM) and, therefore, have pathological consequences like Alzheimer’s disease and multiple cancers. Upon internalization, cargo-laden vesicles released from the PM fuse with the sorting …

Extracellular Mechanotransduction In Marfan Syndrome: An Equivalence Principle, Stephen Haller

Theses & Dissertations

Biological tissues continuously experience mechanical stress and have evolved sophisticated mechanisms to sense mechanical stimuli. While traditional viewpoints regard cells as the ultimate sensors and processors of mechanical information, compounding evidence demonstrates that extracellular matrix, the structural component of tissues, also exhibits evolved molecular responses to force. This led us to propose a new paradigm termed extracellular mechanotransduction, in which matrix orchestrates a complementary form of force integration distinct from traditional cellular and extracellular viewpoints. We thus propose that force-sensitive signaling mechanisms evolved within the extracellular space to help cells maintain mechanical homeostasis in tissues. In this dissertation, we apply …

Paf1/Pd2 Augments Pancreatic Cancer Progression And Mediates Radiation And Chemoresistance, Sanchita Rauth

Theses & Dissertations

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Novel Molecular Mechanisms Of C-Terminal Eps15 Homology Domain (Ehd) Proteins In Endocytic Trafficking And Primary Ciliogenesis, Tyler M. Jones

Theses & Dissertations

Endocytic membrane trafficking is a key cellular process that is critical for regulating the transport of internalized cargoes such as lipids and receptors. Our lab focuses on understanding the mechanisms and cellular functions of the proteins that regulate this pathway. One family of proteins that has seen significant interest over recent years is the C-terminal Eps15 Homology Domain (EHD) family of proteins. Mammalians have four EHD paralogs (EHD1-4) that are expressed ubiquitously in tissues. These proteins have distinct yet overlapping functions in regulating endocytic pathways. EHD1 has been shown to induce constriction and is recruited to induce fission of tubular …

Probing The Role Of Astrocytes In The Pathology Of Fragile X Syndrome With Human Stem Cells, Baiyan Ren

Theses & Dissertations

Fragile X syndrome (FXS) is an X-linked neurodevelopmental disorder related to intellectual disability and the most common monogenic cause of autism spectrum disorder. FXS is mainly caused by an expansion of CGG repeats in the 5’-untranslated region of fragile X mental retardation 1 (FMR1) gene, leading to the loss of expression of fragile X mental retardation protein (FMRP). Astrocytes are the most abundant glial cells in the central nervous system (CNS). Loss of FMRP in astrocytes has been found to contribute to structural and functional synaptic deficits in the Fmr1-KO mouse model. The contribution of human astrocytes, however, to the …

Usp11 And Usp7 Deubiquitinases Regulate Sprtn Auto-Proteolysis And Sprtn-Mediated Dna-Protein Crosslink Repair, Megan C. Perry

Theses & Dissertations

DNA repair pathways that recognize and remove damaged DNA are vital for maintenance of genomic stability and prevention of tumorigenesis. Conversely, these pathways may be robust in tumor cells, thus diminishing the anti-cancer potential of available therapies. DNA-protein crosslinks (DPCs) are particularly deleterious DNA adducts that occur when proteins become irreversibly covalently bound to the DNA. DPCs represent a diverse group of lesions, as any protein can be crosslinked to the DNA duplex by non-specific crosslinking agents like reactive aldehydes and radiation. Additionally, functional DNA-binding proteins such as topoisomerases may become permanently crosslinked to DNA ends by abortive enzymatic processes …

Functional Characterization Of Cancer-Associated Dna Polymerase Ε Variants, Stephanie R. Barbari

Theses & Dissertations

Replicative DNA polymerases ε (Polε) and δ (Polδ) achieve high fidelity DNA synthesis through a precise balance of polymerization and exonucleolytic proofreading. Errors that escape proofreading are corrected by DNA mismatch repair (MMR). Ultramutated human cancers with proficient MMR carry alterations in the exonuclease domain of Polε, which were initially predicted to abolish proofreading. However, functional studies in yeast of the most recurrent Polε-P286R variant suggested defects beyond a loss of exonuclease activity. Indeed, biochemical analysis of the yeast Polε-P286R analog revealed increased polymerization capacity in addition to decreased proofreading, which enables efficient mismatch extension and bypass of replication-blocking non-B …

A Pkcα-Mediated Growth Suppressive Mek-Erk Signaling Axis In Intestinal Epithelial Cells, Navneet Kaur

Theses & Dissertations

Members of the protein kinase C (PKC) family of serine/threonine kinases are involved in regulation of fundamental cellular functions, including proliferation, differentiation, survival, migration, and transformation. Increasing evidence points to anti-proliferative and tumor suppressive role of PKCs. Our laboratory and others have reported that the classical PKC isozyme, PKCαnegatively regulates proliferation and tumorigenesis in the intestinal epithelium. Our laboratory has further determined that PKCα signaling induces a program of cell cycle withdrawal in intestinal epithelial cells that involves downregulation of the pro-proliferative proteins, cyclin D1 and Id1, and upregulation of the cyclin dependent kinase (CDK) inhibitor, p21^Cip1. Unexpectedly, …

Nuclear Receptor Coactivator 3 In Endoplasmic Reticulum Stress And Stress Granule Dynamics In Pancreatic Cancer, Andrew Kisling

Theses & Dissertations

Pancreatic cancer is predicted to be the second-leading cause of cancer-related deaths within the next decade. Nuclear receptor coactivator 3 (NCOA3/SRC3/AIB1) regulates an array of metabolic and signaling pathways and has been established by our group and others as a critical regulator pancreatic cancer progression and metastasis. A recent study demonstrated NCOA3 regulation by the IRE1α-XBP1 axis of the unfolded protein response (UPR), suggesting a link between NCOA3 and cellular stress management. Furthermore, NCOA3 has been shown to directly bind to a scaffolding protein of stress granules (SGs). Since SG assembly is regulated by the UPR, we hypothesized that NCOA3 …

Molecular Mechanisms Of Aberrant Protein Glycosylation In Pancreatic Cancer Stemness And Metastasis, Frank Leon

Theses & Dissertations

A myriad of genetic and other abnormal changes underlies the aggressiveness and dissemination properties observed in pancreatic cancer (PC). Aberrant protein glycosylation is a commonly observed feature in PC. The modification of protein O-glycosylation is mediated by glycosyltransferases, which attach and sequentially elongate monosaccharides on Serine/Threonine (Ser/Thr) motifs. Aberrant glycosylation is recognized as an emerging hallmark of cancer where a disruption in normal glycosylation results in irregular O-glycans.

This dissertation research has investigated the consequences of aberrant protein glycosylation on stemness and enhancement of metastatic properties in pancreatic ductal adenocarcinoma (PDAC). Several publications have reported aberrant O-glycosylation increases in oncogenic …

Dynamics Of Protein-Dna Interactions Characterized By Atomic Force Microscopy, Yaqing Wang

Theses & Dissertations

This thesis describes the nanoscale studies of protein-DNA interactions with different complexities using atomic force microscopy (AFM). One of the systems deals with DNA replication rescue. To maintain the genetic integrity, replication machinery needs to minimize the error rate, repair the damages, and restart the stalled replication caused by the attacks from the environment and inside the cell. The stalled replication rescue is orchestrated by a series of proteins, for example, the DNA helicases PriA and RecG and the ssDNA binding protein (SSB).

We demonstrated that SSB stimulates the restart process in two aspects. First, SSB facilitates the binding of …

Fgfr4 Glycosylation And Processing In Cholangiocarcinoma Promote Cancer Signaling, Andrew J. Phillips

Theses & Dissertations

Cholangiocarcinoma is a cancer of cholangiocytes, or epithelial cells lining the biliary tract. It is associated with a poor prognosis and additional therapeutic treatments are needed to help patients affected by this disease. Fibroblast growth factor receptor 4 (FGFR4) is receptor tyrosine kinase that is involved in various physiologic and pathologic processes. TCGA analysis of thirty different tumor types showed the highest FGFR4 mRNA levels in cholangiocarcinoma. At the protein level, FGFR4 was observed in the majority of cholangiocarcinomas screened and, higher levels were associated with a poorer prognosis. FGFR4 is an N-linked glycosylated receptor tyrosine kinase that we show …

Fibroblast Growth Factor Receptor 4 And R4-Icd Increased Proliferation, Cell Survival And Metastasis In Cholangiocarcinoma, Yamnah Hafeji

Theses & Dissertations

Fibroblast growth factor receptor 4 (FGFR4) is over-expressed in many cancers, including cholangiocarcinoma (CCA). FGFR4 is activated by fibroblast growth factor ligand 19 (FGF19) and plays a critical role in CCA progression. An intracellular cleaved product of FGFR4, referred as R4-ICD (FGFR4 intracellular domain) is also overexpressed in CCA. However, the specific role of R4-ICD in CCA is unknown. In this study, we hypothesized that FGFR4 and R4-ICD play a role in cell proliferation, cell survival and metastasis in CCA. To test this, FGFR4 and R4-ICD were cloned into a cholangiocarcinoma cell line (HuCCT-1) that does not endogenously express FGFR4. …

Design, Synthesis And Evaluation Of Novel Inhibitors Of Type 5 And 10 17Β-Hydroxysteroid Dehydrogenases, Ahmed Morsy

Theses & Dissertations

17β-Hydroxysteroid dehydrogenases (17β-HSDs) are essential enzymes in steroid metabolism. More and more evidence points to the pivotal contributions of these enzymes in various other metabolic pathways. Therefore, the latest research results give new insights into the complex metabolic interconnectivity of the 17β-HSDs with human diseases. This dissertation focuses on the metabolic activities of type 5 and 10 17β-HSDs. More specifically, regarding 17β-HSD5 contributions to the progression of prostate cancer (PCa) and 17β-HSD10 aggravation of amyloid-beta (Aβ)-induced toxicity in Alzheimer's disease (AD).

The second leading cause of cancer-related death in males is PCa, with the highest incidence rate of all cancers …

Secreted Mucin 5ac-Mediated Epithelial And Stromal Modulations Augment Pancreatic Cancer Aggressiveness, Koelina Ganguly

Theses & Dissertations

The mucosal layer that shields the epithelium of the body cavities is made up of high molecular weight, heavily glycosylated proteins called mucins that are broadly categorized into transmembrane and secreted members. Aberrant expression of secreted mucin MUC5AC has been implicated in lung, stomach, and colon cancer pathologies. MUC5AC is expressed de novo in the pancreas upon oncogenic insult, and its abundance in pancreatic tumor and circulation correlates to disease progression. However, few studies have explored beyond the diagnostic and prognostic significance of MUC5AC in pancreatic cancer (PC).

In this dissertation, we sought to investigate the mechanistic contribution of MUC5AC …

Development Of In-Silico Pipelines For Identification And Characterization Of Biomarker Panels And Therapeutic Interventions In Gastro-Intestinal (Gi) Cancers, Pranita Atri

Theses & Dissertations

Gastro-intestinal (GI) malignancies, including gastric, colorectal, and pancreatic cancers, have maintained their high overall mortality due to a lack of prognostic and diagnostic biomarkers and potential therapeutic modalities. While efforts have been made to improve both early detection and therapeutic interventions in these cancers, failure of conventional approaches have proven to be a big challenge, and alternate approaches are needed. Computational biology approaches owing to lesser time and more per target success rate offer a unique solution here. The current study explored the use of computational biology techniques to study the various aspects relating to GI malignancies. First, we sought …

Mucin And Splice Variant Profiles Of Pancreatic Adenocarcinoma Predict Patient Survival And Subtyping, Christopher M. Thompson

Theses & Dissertations

PDAC is a pancreatic epithelial malignancy and demonstrates aggressive progression and bleak patient prognosis. Despite decades of research, the evolution of novel diagnostics and intervention modalities for PDAC is stagnant. This dissertation explores the characteristic aberrant and elevated expression of mucins in PDAC. Beginning with the hypothesis that mucins are associated with disease aggressiveness, analysis of PDAC patient survival in TCGA revealed no associations between single mucin expression and patient survival. This led to the underlying issue of PDAC tumor cellularity since this disease demonstrates variability in the proportion of cancer cells within the tumor. Tumor purity assessed with the …

Role Of Endocytic Machinery Regulators In Egfr Traffic And Viral Entry, Insha Mushtaq

Theses & Dissertations

STUDY 1: Role of endocytic regulator EHD1 and its binding partner RUSC2 in EGFR traffic

Abstract

Epidermal growth factor receptor (EGFR) is a prototype receptor tyrosine kinase and an oncoprotein in many solid tumors. Cell surface display of EGFR is essential for cellular responses to its ligands. While post activation endocytic trafficking of EGFR has been well elucidated, little is known about mechanisms of basal/pre-activation surface display of EGFR. Here, we identify a novel role of the endocytic regulator EHD1 and a potential EHD1 partner, RUSC2, in cell surface display of EGFR. EHD1 and RUSC2 colocalize with EGFR in vesicular/tubular …

Deciphering The Catalytic Mechanism Of Human Manganese Superoxide Dismutase, Jahaun Azadmanesh

Theses & Dissertations

The livelihood of human cells is heavily dependent on the ability to modulate the presence of highly reactive oxygen-based molecules termed reactive oxygen species (ROS). In excess, ROS facilitate oxidative damage to the macromolecules of cellular life. SODs are the major family of antioxidant proteins that prevent the buildup of overwhelming amounts of ROS within cells. Sometimes dubbed the “first line of defense” against oxidative damage, SODs defend against the harmful accumulation of ROS by eliminating superoxide. Superoxide is a ROS itself that is also a precursor to much more harmful ROS molecules. MnSOD is the manganese containing form of …

Identification Of Clonal Evolution Pattern And Mutation Event Associated With Relapsed/Refractory Diffuse Large B-Cell Lymphoma Using Next-Generation Sequencing, Cheng Wang

Theses & Dissertations

Diffuse large B-cell lymphoma (DLBCL) is an aggressive lymphoid malignancy. About 30% of DLBCL cases respond poorly to initial treatment and eventually relapse. For these patients, the current treatment regimen is quite limited, and the prognosis is poor. Gene mutations and genetic alterations play an important role in lymphomagenesis. However, the genetic alterations or gene mutations underlying the disease resistance/relapse in DLBCL are still unknown. The clonal evolution during the process of disease progression is elusive as well. Our goal is to study the genetic alterations in DLBCL, particularly paired diagnostic and relapsed/refractory DLBCL, to better understand the mutation landscape …

Characterizing The Critical Role Of Metabolic And Redox Homeostasis In Colorectal Cancer, Danielle Frodyma

Theses & Dissertations

Metabolic alterations are a hallmark of cancer and the mechanism by which these adaptations sustain cancer cell growth are complex and dependent on tissue type. In colon cancer, Peroxisome Proliferator Activated Receptor γ Coactivator 1 β(PGC1��) and Estrogen-Related Receptor α (ERR��) are overexpressed and contribute to tumor growth. Previous studies have shown that PGC1�� and ERR�� regulate many metabolic processes by controlling vital gene expression. Here, we show that PGC1�� and ERR�� drive oxidative phosphorylation and glycolysis in colon cancer cell lines and we evaluated downstream effectors and processes.

A dysfunction in the reductive and oxidative capacity of the cell …

Mechanisms By Which Mnte-2-Pyp Suppresses Prostate Cancer Cell Growth, Yuxiang Zhu

Theses & Dissertations

Prostate cancer patients are often treated with radiotherapy. MnTE-2-PyP, is a superoxide dismutase (SOD) mimic and a known radioprotector of normal tissues. Our recent work demonstrates that MnTE-2-PyP also inhibits prostate cancer progression with radiotherapy; however, the mechanisms remain unclear. In this thesis, we identified that MnTE-2-PyP-induced intracellular H2O2 levels are critical in inhibiting growth of prostate cancer cells. We found that MnTE-2-PyP induced protein oxidations in PC3 cells and one major group of oxidized protein targets were involved in energy metabolism. The oxidative phosphorylation rates were significantly enhanced in both PC3 and LNCaP cells with MnTE-2-PyP treatment, but mitochondrial …

Elucidating The Role Of Ecdysoneless In Mrna Processing, Irfana Saleem

Theses & Dissertations

The mammalian orthologue of the evolutionarily conserved Ecdysoneless (ECD) protein is required for embryogenesis, cell cycle progression and mitigation of ER stress. However, the molecular mechanisms of ECD function in mammalian cells remain unclear. Here, using mass spectrometry analysis of the mammalian ECD interactome, we identified several components of the mRNA export complexes as binding partners of ECD and then characterized the functional interaction of ECD with key mRNA export-related DEAD BOX protein helicase DDX39A and its associated partners. FISH analysis of Poly-A-tailed mRNAs revealed that ECD depletion/deletion blocks the mRNA export from the nucleus to the cytoplasm. We have …